The present invention relates to methods and compositions for the detection of Bacillus species such as Bacillus anthracis and Bacillus globigii as well as Clostridium perfringens. It relies on nucleic acid sequence differences in spore protein genes carried in the genomic sequence of these organisms.
The genus Bacillus is composed of rod-shaped, gram-positive, aerobic or (under some conditions) anaerobic bacteria widely found in soil and water. Most strains of Bacillus are not pathogenic for humans and only infect them incidentally in their role as soil organisms; a notable exception is Bacillus anthracis, which causes anthrax in humans and domestic animals.
In addition to its role as a naturally occurring pathogen, Bacillus anthracis may also be used as a biological weapon. Because Bacillus organisms are widely distributed in the environment, and because they are very closely related genetically, there is need for a reliable method to distinguish species members in various types of samples.
Considerable efforts have been made to develop sensitive, species specific tests for Bacillus microorganisms with only limited results. Most of the Bacillus anthracis PCR detection systems have targeted plasmid sequences, yet mobility of these genetic elements make them an unreliable detection target. Several of the PCR type assays to genomic sequences target non-gene-coding sequences, which often proves not to correlate well with species identity. The weaknesses in each of these systems underscores the need for a more reliable Bacillus identification method.
The subject invention provides novel methods and materials for specifically identifying certain rod-shaped, gram-positive bacteria. These methods and materials rely on the discovery that certain small acid-soluble protein (sasp) gene targets are present in the bacterial coats of Bacillus and Clostridium organisms. These sasp gene targets may be identified by a technique described here as xe2x80x9cheterologous PCRxe2x80x9d. More specifically, these methods and materials provide means for specifically identifying certain members of the Bacillus genus, especially Bacillus anthracis and Bacillus globigii, and members of the Clostridium genus, especially Clostridium perfringens. Using heterologous PCR, novel gene targets are identified that yield species-specific regions for detection and identification. Based on this technique, assay techniques for identifying the organisms Bacillus anthracis, Bacillus globigii and Clostridium perfringens have been developed.
Another aspect of the invention comprises use of a small acid soluble protein (sasp-B) gene sequence in Bacillus anthracis that contains a specific region of stable nucleotide insertion absent in even the closest Bacillus relatives. This unique biomarker is a probe-binding region for the specific detection of Bacillus anthracis. Additional regions of sequence within the gene were used to develop a Bacillus anthracis specific amplification and detection system. The system is capable of distinguishing Bacillus anthracis from near neighbors during two phases of the amplified assay: during the amplification reaction, and by probe hybridization to the Bacillus anthracis specific biomarker within the amplified product for sequence confirmation.
As another aspect of the invention, the present Bacillus anthracis detection system either alone, or multiplexed with one or more additional genomic and/or virulence plasmid markers, is of use as a diagnostic or confirmatory tool in Public Health and clinical laboratories, as well as in the law enforcement sector. The system may be adapted to a variety of amplification and detection platforms in order to accommodate the technical and fiscal capabilities of the laboratory, or used in xe2x80x98fieldxe2x80x99 settings.
As yet another aspect of the invention, building upon the discovery that small acid-soluble spore protein genes maintain species-specific sequence signatures, analogous regions were identified among the sasp of Bacillus globigii (which is used as a non-pathogenic xe2x80x98surrogatexe2x80x99 for Bacillus anthracis in research and development applications, defense, and emergency response modeling, etc.). Taking advantage of this discovery, Bacillus globigii specific PCR was designed and reduced to practice.
Another aspect of the invention is to provide a two-step amplification/detection system. A particular sasp gene is selected for amplification, and its identity determined by a species-specific probe. Although the present invention is described in detail in connection with a PCR, it is understood that, based on the present teachings, other amplification and/or identification systems could be devised.